There is strong evidence that a prophylactic HIV vaccine will have to mobilize a combination of high quality cellular and humoral mediated immune responses. Such an approach will require strong HIV-specific CD4 T cell responses to provide help to both arms of the immune system. However, in contrast to HIV-specific CD8 T cells, little is known about the function of HIV- specific CD4 T cells and how they contribute to control HIV infection. More importantly, the functional characteristics and the impact of HIV-specific T follicular helper (Tfh) cells on the production of broadly neutralizing antibody responses are largely unknown. It is now evident that only a small fraction of HIV infected subjects develop broadly neutralizing antibody responses capable of neutralizing a wide range of viruses. Understanding the molecular basis of what constitutes an effective HIV-specific CD4 T cell response associated with the development of broadly neutralizing antibody responses in those subjects will provide invaluable information for the rational design of vaccine approaches. We propose to use a systems biology approach that will allow unbiased, comprehensive characterization of HIV-specific CD4 T cells not only from peripheral blood but also from lymph nodes, the main anatomic compartment of Tfh cells. This will provide crucial information on the intrinsic characteristics of HIV-specific Tfh cells that are able to orchestrate functional humoral responses against HIV. In Aim 1, we will use next generation sequencing (NGS) to define the transcriptional signatures of tetramer sorted HIV- specific CD4 T cells that are associated with the development of broadly neutralizing antibody responses. We will use this approach to identify the transcriptional signatures of HIV-specific Tfh cells from lymph nodes. We will also perform high throughput characterization of HIV- specific CD4 T cells at the single cell level using state-of-the-art mass cytometry and a novel flow-FISH assay (fluorescent in vitro hybridization) in order to characterize the subpopulations that express markers identified to be associated with superior CD4 help to B cells. In Aim 2, we will identify the microRNome of HIV-specific CD4 T cells in blood and lymph nodes to investigate the role of microRNAs in the post-translational regulation of HIV-specific CD4 T helper functions that are associated with the development of neutralizing antibodies and functional germinal center formation. Combining transcriptional signatures with miRNA profiles using state-of-the-art bioinformatics approaches will allow for the dissection of molecular networks that cannot be understood by traditional research strategies. In Aim 3, we propose to investigate immunotherapeutic interventions to redirect HIV-specific CD4 T cells towards optimal CD4 help. We will manipulate extrinsic (cytokines) and intrinsic (transcription factors and miRNAs) factors identified in Aims 1 and 2 to improve CD4 help to B cells. The proposed work will shed light on the molecular networks that regulate HIV-specific CD4 T cell help to B cells for the development of broadly neutralizing antibody responses and will investigate novel interventions to redirect CD4 T cell responses towards a desired immune response.